This proposal describes exploratory research into the secretion and bacterial association of heat-labile enterotoxin (LT) produced by enterotoxigenic E. coli (ETEC). ETEC is an important diarrheagenic pathogen in third world countries and is a biodefense threat since military personel are likely to acquire this pathogen from non-sterile water and food sources in the field. This research laboratory has discovered that LT binds lipopolysaccharide (LPS) on the bacterial surface and on the surface of secreted outer membrane vesicles via an interaction between the B subunit of LT (LTB) and the Kdo core of LPS. The described study will elucidate the site for LPS binding on LTB using site-directed mutagenesis based on the likely binding site for the oligosaccharide. In addition, binding studies will reveal the affinity between LTB and components of LPS. The relationship of LT binding to LPS and toxicity will be investigated. The bacterial localization of virulence factors may influence their role in pathogenesis. Therefore, fluorescence microscopy and tagging strategies will be used to investigate the bacterial surface localization of secreted, bound LT. Since cholera toxin (CT) secreted by Vibrio cholera is highly homologous to LT and was also determined to bind to E. coli LPS, LT and CT will be compared in binding and localization studies. The data resulting from these aims will open new avenues of research regarding lectin binding sites, the localization of LT, and the difference in the enteric toxicity caused by ETEC and V. cholerae.